Linear microarrays

ABSTRACT

The present invention provides a method and a composition for detecting the levels of a plurality of biomolecular probes in a sample. In particular, the invention relates to a hybridization composition for detecting the presence or levels of different polynucleotide sequences in a sample.

FIELD OF THE INVENTION

The present invention provides a method and a composition for detectingthe levels of a plurality of biomolecular probes in a sample.

BACKGROUND OF THE INVENTION

DNA-based arrays can provide a simple way to explore the expression of asingle polymorphic gene or a large number of genes. When the expressionof a single gene is explored, DNA-based arrays are employed to detectthe expression of specific gene variants. For example, a p53 gene arrayis used to determine whether individuals are carrying mutations thatpredispose them to cancer. Alternatively, a P450 gene array is used todetermine whether individuals have one of a number of specific mutationsthat could result in increased drug metabolism, drug resistance or drugtoxicity.

DNA-based array technology is especially relevant to the rapid screeningof expression of a large number of genes. There is a growing awarenessthat gene expression is affected in a global fashion. A geneticpredisposition, disease or therapeutic treatment may affect, directly orindirectly, the expression of a large number of genes. In some cases arelationship between a disease or therapeutic treatment and theexpression of particular genes may be expected, such as where the genesare known to be part of a signaling pathway implicated in a disease. Inother cases, such as when the genes' function is unidentified or thegenes participate in separate signaling pathways, the relationshipbetween a disease and particular genes may be unexpected.

Therefore, DNA-based arrays can be used to investigate how geneticpredisposition, disease, or therapeutic treatment may affect theexpression of individual genes or a group of genes.

SUMMARY OF THE INVENTION

The present invention provides a hybridization composition for detectingthe levels of a plurality of biomolecular probes in a sample. Thecomposition comprises (a) a capillary-like casing; and (b) a substrateimmobilized in said casing. The substrate's surface contains a pluralityof regions arranged in a defined manner with respect to the length ofsaid casing and each of the regions has one or more differentimmobilized target. Additionally, the substrate's surface is in closeproximity with the inner surface of said casing so as to minimize theratio of liquid volume contained within said casing to the substrate'ssurface area. The linear density of the plurality of defined regions isgreater than 1×10³/cm, preferably is greater than 1×10³/cm, and morepreferably is greater than 1×10⁶/cm.

In one preferred embodiment, the biomolecular probes comprisepolynucleotide probes in a sample and the targets are complementarypolynucleotide sequences. In another preferred embodiment, the ratio ofliquid volume contained within said casing to hybridization surface areais less than about 1×10⁻⁵ m, preferably less than 1×10⁻⁷ m, morepreferably less than 1×10−⁹m. In yet another preferred embodiment, thesubstrate is a plurality of beads whose diameters approximate the innerdiameter of the capillary-like casing. In a another preferredembodiment, the substrate is a rod whose diameter approximates the innerdiameter of the capillary-like casing. Typically, the diameter of thesubstrate is more than 90%, preferably more than 95%, of the innerdiameter of the capillary-like casing. In a further embodiment, thesubstrate is an agarose s plug flush against the inner surface of thecapillary-like casing.

The present invention also provides a method for detecting the levels ofa plurality of biomolecular probes in a sample. The method comprises (i)contacting the sample comprising the plurality of biomolecular probeswith a hybridization composition comprising (a) a capillary-like casingand (b) a substrate immobilized in said casing under conditionseffective to form hybridization complexes between biomolecular probesand immobilized targets; and (ii) detecting the hybridization complexes.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 illustrates a cross-sectional view of one embodiment of theinvention, where the hybridization composition comprises a substratecomprising a plurality of beads.

FIG. 2 illustrates a cross-sectional view of a second embodiment of theinvention, where the hybridization composition comprises a substratecomprising a plurality of defined regions perpendicular to the length ofa rod.

FIG. 3 illustrates a cross-sectional view of a third embodiment of theinvention, where the hybridization composition comprises a substratecomprising a plurality of defined regions parallel to the length of arod.

FIG. 4 illustrates a cross-sectional view of yet another embodiment ofthe present invention, wherein the invention comprises a plurality ofhybridization compositions.

DESCRIPTION OF THE INVENTION

The present invention provides a hybridization composition which isuseful in hybridization reactions where high throughput hybridizationexperiments utilizing small sample volumes are desired. The compositionallows for hybridization experiments to be performed with sample volumesthat are substantially less than those used today in microarrayhybridization experiments. Typically, in experiments usingtwo-dimensional microarrays, volumes for sample delivery, hybridizationor washes are required in the range of 12 to 200 microliters. Incontrast, when the present invention is employed, volumes in the rangeof 10 nanoliters to 10 microliters are necessary for sample delivery,hybridization or washes.

The invention is a composition comprising one or more capillary-likecasings with one or more input openings and one or more output openingsand a substrate immobilized in each of said capillary-like casings. Akey feature of the invention is that the substrate's surface is in closeproximity with the inner surface of the capillary casing so as tominimize the ratio of liquid volume contained within said casing to thesubstrate's surface area. In this manner the volume for sample deliveryand hybridization reactions is minimized. Typically, for two-dimensionalmicroarrays the ratio of sample volume to surface area is about 1×10−⁵m.In contrast, the present invention provides for ratios of sample volumeto hybridization surface area less than 1×10⁻⁵m, preferably less than1×10−⁷m, and more preferably less than 1×10⁻⁷ m. The ratios can bevaried by changing, for example, the inner diameter of thecapillary-like casing or the surface area of the substrate.

One preferred embodiment of the present invention is illustrated in FIG.1. In this embodiment, the capillary-like casing is a capillary tube 2and the substrate comprises a plurality of beads, such as bead 4. Eachbead has at least one type of immobilized target, such as immobilizedtarget 6, on its surface. A key feature of the invention is that eachbead is localized in defined locations along the length of the casing,such that the hybridization composition is defined by having a lineardensity of different polynucleotide sequences of at least 1/cm,preferably at least 1×10³/cm, more preferably greater than 1×10⁶/cm.Additionally, the inner diameter of the capillary and the diameter ofthe bead are selected to be substantially similar. This means thediameter of the bead is more than 90%, preferable more than 95%, andmore preferably more than 98%, the length of the inner diameter of thecapillary-like casing. Samples, prehybridization buffers, hybridizationbuffers, and washes flow into the hybridization composition througheither first 8 or second 10 open ends and flow out, preferably, throughthe opposite end.

A second preferred embodiment is illustrated in FIG. 2. In this secondembodiment, the capillary-like casing is a capillary tube 12 and thesubstrate comprises a rod 14 coextensive with the capillary tube. Therod may comprise bands, such as band 16 containing at least one type ofimmobilized target, such as target 18. Band regions containing differentimmobilized targets may alternate with bands lacking a target, such asunmodified band 20. Again, the hybridization composition is defined byhaving a linear density of different polynucleotide sequences of atleast 1/cm, preferably at least 1×10³/cm, more preferably greater than1×10⁶/cm. Additionally, the inner radius of the capillary tube and theouter radius of the rod ;re substantially similar. Samples,prehybridization buffers, hybridization buffers, or washes flow into thehybridization composition through either first 22 or second 24 open endsand flow out, preferably, through the opposite end. Alternatively, thesubstrate may comprise alternating agarose plugs modified by at leastone target and unmodified agarose plugs.

A third embodiment is illustrated in FIG. 3. In this instance, thecapillary-like casing is a capillary tube 30 and the substrate comprisesa rod 32 coextensive with the capillary tube. In this case, however,instead of having defined regions, such as defined region 34, runningperpendicular to the length of the casing, the discrete regions run inparallel to the length of the casing. Again, each defined region maycontain one or more immobilized targets, such as target 36.Alternatively, the substrate may comprise a plurality of rods containingdifferent polynucleotide sequences in defined regions along the lengthof the casing.

FIG. 4 illustrates how the present invention is implemented in a formatwhere a plurality of hybridization compositions are employed at the sametime. As shown in FIG. 4, three hybridization compositions, such ashybridization composition 40, is connected to one or more neighboringhybridization compositions to form a two-dimensional arrangement 42.Alternatively, the hybridization compositions can be in athree-dimensional arrangement. Connections, such as valve 44, betweenneighboring hybridization compositions 40 and 46 can be in an open orclosed position. When connections are in a closed position, differenthybridizing compositions can be subjected to different samples and/orhybridizing conditions. The dimensions of the capillary-like casing, thesubstrate, the defined regions or the immobilized targets are not drawnto scale in FIGS. 1 through 4.

The capillary-like casings are preferably capillaries, cylinders and thelike and are preferably optically transparent. Additionally, the casingspreferably have low autofluorescence. The substrate may be porous,solid, rigid, corrugated, layered or semi-rigid. The substrate ispreferably optically transparent and has a low autofluorescence.Preferably, the substrate comprises beads or rods or plugs of 1 mm indiameter, preferably 100 microns, more preferably less than 10 microns,and even more preferably less than 1 micron in diameter.

For example, as illustrated in Table 1, if beads with a radius of 5×10⁷m (diameter=1 micron) are selected and the inner radius of the capillaryis about 5% larger than the bead radius, then the ratio of hybridizationvolume to surface area can be reduced to 1.09×10⁷ m.

TABLE 1 Bead Bead Capillary Hybridization radius surface interiorHybridization volume/Surface (m) area (m²) volume (m³) volume (m³) area(m) 5 × 10⁻⁷ 3.14 × 10⁻¹² 3.42 × 10⁻¹⁵ 8.66 × 10⁻¹⁹ 1.09 × 10⁻⁷ 5 × 10⁻⁶3.14 × 10⁻¹⁰ 3.42 × 10⁻¹² 8.66 × 10⁻¹⁶ 1.09 × 10⁻⁶ 5 × 10⁻⁵ 3.14 × 10⁻⁸ 3.42 × 10⁻⁹  8.66 × 10⁻¹³ 1.09 × 10⁻⁵ 5 × 10⁻⁴ 3.14 × 10⁻⁶  3.42 × 10⁻⁶ 8.66 × 10⁻¹⁰ 1.09 × 10⁻⁴

As illustrated in Table 2, if an inner rod with a radius 5×10⁷ m(diameter=1 micron) is selected and the inner radius of the capillary isabout 5% larger than the rod radius, then the ratio of hybridizationvolume to surface area can be reduced to 2.56×10⁸ m.

TABLE 2 Inner Inner capillary capillary Capillary Hybridization radiussurface interior Hybridization volume/Surface (m) area (m²) volume (m³)volume (m³) area (m) 5 × 10⁻⁷ 3.14 × 10⁻¹² 8.05 × 10⁻¹⁶ 8.66 × 10⁻¹⁹2.56 × 10⁻⁸ 5 × 10⁻⁶ 3.14 × 10⁻¹⁰ 8.05 × 10⁻¹³ 8.66 × 10⁻¹⁶ 2.56 × 10⁻⁷5 × 10⁻⁵ 3.14 × 10⁻⁸  8.05 × 10⁻¹⁰ 8.66 × 10⁻¹³ 2.56 × 10⁻⁶ 5 × 10⁻⁴3.14 × 10⁻⁶  8.05 × 10⁻⁷  8.66 × 10⁻¹⁰ 2.56 × 10⁻⁵

The substrates can be coated or bonded with a polymeric film or apolymer layer. In one embodiment, a glass rod is coated evenly with asubstance that gives the slide an even binding surface that ispositively charged, such as amino silane or polylysine. In anotherembodiment, a substrate is coated with a polymer layer which contributesreactive groups, such as epoxide groups, for chemical coupling ofpolynucleotide sequences.

Immobilized on a plurality of defined regions of the substrate'ssurface, are localized multiple copies of one or more polynucleotidesequences, preferably copies of a single polynucleotide sequence. Apolynucleotide refers to a chain of nucleotides. Preferably, the chainhas from 5 to 10,000 nucleotides, more preferably from 15 to 3,500nucleotides.

The plurality of defined regions on the substrate can be arranged in avariety of formats. For example, the regions may be arrangedperpendicular or in parallel to the length of the casing. Theseimmobilized copies of a polynucleotide sequence are suitable for use asa target polynucleotide in hybridization experiments. Furthermore, theprobes do not have to be directly bound to the substrate, but rather canbe bound to the substrate through a linker group. The linker groups maytypically vary from about 6 to 50 atoms long. Preferred linker groupsinclude ethylene glycol oligomers, diamines, diacids and the like.Reactive groups on the substrate surface react with one of the terminalportions of the linker to bind the linker to the substrate. The otherterminal portion of the linker is then functionalized for binding thepolynucleotides.

To prepare beads coated with immobilized polynucleotide sequences, beadsare immersed in a solution containing the desired polynucleotidesequence and then immobilized on the beads by covalent or noncovalentmeans. Alternatively, when the polynucleotides are immobilized on rods,a given polynucleotide can be spotted at defined regions of the rod.Typical dispensers include a micropipette delivering solution to thesubstrate with a robotic system to control the position of themicropipette with respect to the substrate. There can be a multiplicityof dispensers so that reagents can be delivered to the reaction regionssimultaneously. In one embodiment, a microarray is formed by usingink-jet technology based on the piezoelectric effect, whereby a narrowtube containing a liquid of interest, such as oligonucleotide synthesisreagents, is encircled by an adapter. An electric charge sent across theadapter causes the adapter to expand at a different rate than the tubeand forces a small drop of liquid onto a substrate (Baldeschweiler etal. PCT publication WO95/251116).

Samples may be any sample containing polynucleotides (polynucleotideprobes) of interest and obtained from any bodily fluid (blood, urine,saliva, phlegm, gastric juices, etc.), cultured cells, biopsies, orother tissue preparations. DNA or RNA can be isolated from the sampleaccording to any of a number of methods well known to those of skill inthe art. For example, methods of purification of nucleic acids aredescribed in Laboratory Techniques in Biochemistry and MolecularBiology: Hybridization With Nucleic Acid Probes. Part I. Theory andNucleic Acid Preparation, P. Tijssen, ed. Elsevier (1993). In. apreferred embodiment, total RNA is isolated using the TRIzol total RNAisolation reagent (Life Technologies, Inc., Rockville, Md.) and RNA isisolated using oligo d(T) column chromatography or glass beads. Afterhybridization and processing, the hybridization signals obtained shouldreflect accurately the amounts of control target polynucleotide added tothe sample.

Sample polynucleotides may be labeled with one or more labeling moietiesto allow for detection of hybridized probe/target polynucleotidecomplexes. The labeling moieties can include compositions that can bedetected by spectroscopic, photochemical, biochemical, bioelectronic,immunochemical, electrical, optical or chemical means. The labelingmoieties include radioisotopes, such as ³²P, ³³P or ³⁵S,chemiluminescent compounds, labeled binding proteins, heavy metal atoms,spectroscopic markers, such as fluorescent markers and dyes, magneticlabels, linked enzymes, mass spectrometry tags, spin labels, electrontransfer donors and acceptors, biotin, and the like.

Labeling can be carried out during an amplification reaction, such aspolymerase chain reaction and in vitro or in vivo transcriptionreactions. Alternatively, the labeling moiety can be incorporated afterhybridization once a probe-target complex his formed. In one preferredembodiment, biotin is first incorporated during an amplification step asdescribed above. After the hybridization reaction, unbound nucleic acidsare rinsed away so that the only biotin remaining bound to the substrateis that attached to target polynucleotides that are hybridized to thepolynucleotide probes. Then, an avidin-conjugated fluorophore, such asavidin-phycoerythrin, that binds with high affinity to biotin is add

Hybridization causes a polynucleotide probe and a complementary targetto form a stable duplex through base pairing. Hybridization methods arewell known to those skilled in the art Stringent conditions forhybridization can be defined by salt concentration, temperature, andother chemicals and conditions. Varying additional parameters, such ashybridization time, the concentration of detergent (sodium dodecylsulfate, SDS) or solvent (formamide), and the inclusion or exclusion ofcarrier DNA, are well known to those skilled in the art. Additionalvariations on these conditions will be readily apparent to those skilledin the art (Wahl, G. M. and S. L. Berger (1987) Methods Enzymol.152:399-407; Kimmel, A. R. (1987) Methods Enzymol. 152:507-511; Ausubel,F. M. et al. (1997) Short Protocols in Molecular Biology, John Wiley &Sons, New York, N.Y.; and Sambrook, J. et al. (1989) Molecular Cloning,A Laboratory Manual, Cold Spring Harbor Press, Plainview, N.Y.).

In a preferred embodiment, hybridization is performed with buffers, suchas 5 ×SSC with 0.1% SDS at 25° C. Subsequent washes may be performed athigher stringency with buffers, such as 0.5×SSC with 0.1% SDS at 25° C.Stringency can also be increased by adding agents such as formamide.Background signals can be reduced by the use of detergent, such as SDSor Triton x-100, or a blocking agent, such as salmon sperm DNA or bovineserum albumin.

Hybridization reactions can be performed in absolute or differentialhybridization formats. In the absolute hybridization format,polynucleotide probes from one sample are hybridized to the targetsequences and signals detected after hybridization complex formationcorrelate to polynucleotide probe levels in a sample. In thedifferential hybridization format, the differential expression of a setof genes in two biological samples is analyzed. For differentialhybridization, polynucleotide probes from both biological samples areprepared and labeled with different labeling moieties. A mixture of thetwo labeled polynucleotide probes is added to a microarray. Afterhybridization and washing, the microarray is examined under conditionsin which the emissions from the two different labels are individuallydetectable. Probes in the microarray that are hybridized tosubstantially equal numbers of target polynucleotides derived from bothbiological samples give a distinct combined fluorescence (Shalon et al.PCT publication WO95/35505). In a preferred embodiment, the labels arefluorescent labels with distinguishable emission spectra, such asCy3/Cy5 fluorophores (Amersham Pharmacia Biotech, Piscataway, N.J.).After hybridization, the microarray is washed to remove nonhybridizednucleic acids and complex formation between the hybridizable arrayelements and the target polynucleotides is detected.

Methods for detecting complex formation are well known to those skilledin the art. In a preferred embodiment, the polynucleotide probes arelabeled with a fluorescent label and measurement of levels and patternsof complex formation is accomplished by fluorescence microscopy,preferably confocal fluorescence microscopy. An argon ion laser excitesthe fluorescent label, emissions are directed to a photomultiplier andthe amount of emitted light detected and quantitated. The detectedsignal should be proportional to the amount of probe/targetpolynucleotide complex at each position of the microarray. Thefluorescence microscope can be associated with a computer-driven scannerdevice to generate a quantitative two-dimensional image of hybridizationintensities. The scanned image is examined to determine theabundance/expression level of each hybridized target polynucleotide.

In a differential hybridization experiment, polynucleotide probes fromtwo or more different biological samples are labeled with two or moredifferent fluorescent labels with different emission wavelengths.Fluorescent signals are detected separately with differentphotomultipliers set to detect specific wavelengths. The relativeabundances/expression levels of the target polynucleotides in two ormore samples is obtained. Typically, microarray fluorescence intensitiescan be normalized to take into account variations in hybridizationintensities when more than one microarray is used under similar testconditions. In a preferred embodiment, individual polynucleotideprobe/target complex hybridization intensities are normalized using theintensities derived from internal normalization controls contained oneach microarray.

It is understood that this invention is not limited to the particularmethodology, protocols, and reagents described, as these may vary. It isalso understood that the terminology used herein is for the purpose ofdescribing particular embodiments only, and is not intended to limit thescope of the present invention which will be limited only by theappended claims. The examples below are provided to illustrate thesubject invention and are not included for the purpose of limiting theinvention.

EXAMPLES Example 1 Preparation of Epoxide Derivatized Agarose

Ten g of agarose (Life Technologies, Inc., Rockville, Md.)) was addedinto a 250 ml round bottom flask with a stirring bar. Forty mls oftoluene and ten 10 mls of 3-glycidoxypropyl-trimethoxysilane(Sigma-Aldrich Corporat was stirred for 10 minutes. 1 ml ofN,N-diisopropylethylamine (Sigma-Aldrich Corporation) was added by a 5ml syringe and the resulting mixture was stirred and heated (85° C.) for65 hours. The reaction flask was then cooled to room temperature. Theepoxide coated agarose was washed with 100ml of toluene followed bywashes with 100 ml of ethanol, then 100ml of hexane, then with 100 ml ofacetone. The epoxide derivatized agarose was dried under the vacuum for2 hours and stored in a desiccator prior to use. A similar procedure wasused to derivatize glass beads.

Example 2 Preparation of Agarose Plug Array

A YP3 59mer labeled at the 3′-end with a Cy3 fluorescent. dye waspurchased from Operon Technologies, Alameda, Calif. The modifiedpolynucleotide was dissolved in water at a concentration of 150 uM. 10ul of the polynucleotide solution vas mixed with 10 mg of the epoxidederivatized agarose. 20 ul of water was added and the resulting slurrywas heated at 40 ° C. for 20 minutes. 970 ul of 1 M MOPS was added andthe slurry boiled until the agarose dissolved. The agarose was thenpoured onto a surface and allowed to set. The agarose sheet was then cutinto bands. Alternating bands of this DNA linked agarose and plain 1%agarose were then plugged into capillaries. The resulting agarose plugarray was about 1 mm in diameter and 5 cm long. The array containedbands of different DNA species that were 2 to 4 mm long. One or morecapillaries were then glued to glass slides for ease of handling.

Example 3 Preparation of Glass Bead Array

The same modified polynucleotide was employed. The polynucleotide wasdissolved in water at a concentration of 150 uM and further diluted to0.5 mg/ml in 0.1% SDS. Epoxide- coated glass beads were immersed in thissolution, mixed by vortexing, and then poured out on a petri dish. Beadswere allowed to dry overnight. They were then washed once with 5×SSC/0.1% SDS, once with 0.5×SSC/0.1% SDS, and once with 0.1% SDS. Beadswere dried under vacuum. A capillary tube with a 0.525 mm radius waspacked with the 0.5 mm beads separated by alternating unmodified beads.In order to keep the beads from falling out of the capillary, plasticshims (pipette tips, purchased from E&K) were inserted into the ends.Capillaries were then glued to glass slides for ease of handling.

Example 4 Hybridization Using the Agarose Plug Array

The array, attached to its glass slide, was immersed in anelectrophoresis chamber filled with 1M MOPS with the capillary parallelto the electric field. One microliter of a 400 uM solution of aCy5-5′-labeled 59mer was added to the end of an agarose pluggedcapillary. The sequence of this second polynucleotide was complementaryto the agarose-immobilized polynucleotide.

A potential of 40 volts was applied to the chamber for two hours. Overthis time the Cy5-labeled polynculeotide was observed to removed fromthe chamber and scanned with a confocal fluorescence microscope using aCy3 and Cy5 detection system. The Cy5-labeled polynucleotide was foundto localize only in regions where complementary Cy3-labeledpolynucleotide was immobilized.

Example 5 Hybridization Using the Glass Bead Array

100 ul of a 4 uM Cy5-5′labeled 59mer in 5×SSC,0.1%SDS was used to fill abead capillary array. The array was then incubated for 2 hours at 60° C.It was then washed in succession with 200 ul 5×SSC, 0.1% SDS, 200 ul0.5×SSC, 0.1%SDS, and 200 ul 0.1%SDS. The array was then dried undervacuum and scanned with a confocal fluorescence microscope using a Cy3and Cy5 detection system. The Cy5-labeled polynucleotide was found tolocalize in regions where complementary Cy3-polynucleotide derivatizedigarose was present.

What is claimed is:
 1. A device for detecting a plurality ofbiomolecular probes in a sample, said device comprising a vesselcomprising: (a) a casing having an inner surface and input and outputopenings, (b) a nonporous substrate having an outer surface andcontained within said casing, (c) a liquid sample comprisingbiomolecular probes and contained within said casing, wherein thesubstrate outer surface contains a plurality of regions arranged in adefined manner and each of said regions has a different one of aplurality of immobilized specific binding targets which specificallybind a corresponding one of the plurality of the biomolecular probes,and said substrate outer surface is in close proximity with the innersurface of said casina so as to minimize the ratio of the liquid samplevolume contained within said casing to the substrate outer surface area,yet permit flow of the liquid sample from the input opening, between thesubstrate outer surface and the casing inner surface, and to the outputopening.
 2. The device of claim 1, wherein the ratio of the liquidsample volume contained within the casing to the substrate outer surfacearea is less than about 1×10⁻⁵ m.
 3. The device of claim 1, wherein theratio of the liquid sample volume contained within the casing to thesubstrate outer surface area is less than about 1×10⁻⁷ m.
 4. The deviceof claim 1, wherein the ratio of the liquid sample volume containedwithin the casing to the substrate outer surface area is less than about1×10⁻⁹ m.
 5. The device of claim 1, wherein the substrate is elongate,and has a length and the regions are arranged with a linear density ofgreater than 1×10³ regions per centimeter of the length.
 6. The deviceof claim 1, wherein the substrate is elongate and has a length and theregions are arranged with a linear density of greater than 1×10⁶regionsper centimeter of the length.
 7. The device of claim 1, wherein thesubstrate is an elongate rod.
 8. The device of claim 1, wherein thesubstrate is an elongate rod less than 1 mm in diameter.
 9. The deviceof claim 1, wherein the substrate is an elongate rod less than 10 um indiameter.
 10. The device of claim 1, wherein the substrate is aplurality of beads.
 11. The device of claim 1, wherein the substrate isa plurality of beads, each bead less than 1 mm in diameter.
 12. Thedevice of claim 1, wherein the substrate is a plurality of beads, eachbead less than 10 um in diameter.
 13. The device of claim 1, wherein thesubstrate is made of glass, optionally surface coated with a polymer topromote immobilization of the binding targets.
 14. The device of claim1, wherein the casing is made of glass.
 15. The device of claim 1,wherein the casing is a capillary tube.
 16. The device of claim 1,wherein said vessel is one of a plurality of vessels in fluidconnection.
 17. The device of claim 1, wherein the immobilized bindingtargets and corresponding biomolecular probes comprise polynucleotideswhich specifically bind by hybridization.
 18. The device of claim 1,wherein the ratio of the liquid sample volume contained within thecasing to the substrate outer surface area is less than about 1×10⁻⁵ m,and the immobilized binding targets and corresponding biomolecularprobes comprise polynucleotides which specifically bind byhybridization.
 19. The device of claim 1, wherein the ratio of theliquid sample volume contained within the casing to the substrate outersurface area is less than about 1×10⁻⁷ m, and the immobilized bindingtargets and corresponding biomolecular probes comprise polynucleotideswhich specifically bind by hybridization.
 20. The device of claim 1,wherein the ratio of the liquid sample volume contained within thecasing to the substrate outer surface area is less than about 1×10⁻⁵m,the substrate is elongate and has a length and the regions are arrangedwith a linear density of greater than 1×l0 ³ regions per centimeter ofthe length, and the immobilized binding targets and correspondingbiomolecular probes comprise polynucleotides which specifically bind byhybridization.
 21. The device of claim 1, wherein the ratio of theliquid sample volume contained within the casing to the substrate outersurface area is less than about 1×10⁻⁷ m, the substrate is elongate andhas a length and the regions are arranged with a linear density ofgreater than 1×10³ regions per centimeter of the length, and theimmobilized binding targets and corresponding biomolecular probescomprise polynucleotides which specifically bind by hybridization. 22.The device of claim 1, wherein the ratio of the liquid sample volumecontained within the casing to the substrate outer surface area is lessthan about 1×10⁵ m, the immobilized binding targets and correspondingbiomolecular probes comprise polynucleotides which specifically bind byhybridization, and the substrate is made of glass, optionally surfacecoated with a polymer to promote immobilization of the binding targets.23. The device of claim 1, wherein the ratio of the liquid sample volumecontained within the casing to the substrate outer surface area is lessthan about 1×10⁻⁷ m, the immobilized binding targets and correspondingbiomolecular probes comprise polynucleotides which specifically bind byhybridization, and the substrate is made of glass, optionally surfacecoated with a polymer to promote immobilization of the binding targets.24. The device of claim 1, wherein the ratio of the liquid sample volumecontained within the casing to the substrate outer surface area is lessthan about 1×10⁻⁵ m, the substrate is elongate and has a length and theregions are arranged with a linear density of greater than 1×10³ regionsper centimeter of the length, the immobilized binding targets andcorresponding biomolecular probes comprise polynucleotides whichspecifically bind by hybridization and the substrate is made of glass,optionally surface coated with a polymer to promote immobilization ofthe binding targets.
 25. The device of claim 1, wherein the ratio of theliquid sample volume contained within the casing to the substrate outersurface area is less than about 1×10⁻⁷ m, the substrate is elongate andhas a length and the regions are arranged with a linear density ofgreater than 1×10³ regions per centimeter of the length, the immobilizedbinding targets and corresponding biomolecular probes comprisepolynucleotides which specifically bind by hybridization and thesubstrate is made of glass, optionally surface coated with a polymer topromote immobilization of the binding targets.
 26. A method fordetecting biomolecular probes in a sample, said method comprising thestep of using a device according to claim 1 to detect specific bindingof the biomolecular probes to the immobilized targets.
 27. A method fordetecting biomolecular probes in a sample, said method comprising thestep of using a device according to claim 2 to detect specific bindingof the biomolecular probes to the immobilized targets.
 28. A method fordetecting biomolecular probes in a sample, said method comprising thestep of using a device according to claim 3 to detect specific bindingof the biomolecular probes to the immobilized targets.
 29. A method fordetecting biomolecular probes in a sample, said method comprising thestep of using a device according to claim 4 to detect specific bindingof the biomolecular probes to the immobilized targets.
 30. A method fordetecting biomolecular probes in a sample, said method comprising thestep of using a device according to claim 5 to detect specific bindingof the biomolecular probes to the immobilized targets.
 31. A method fordetecting biomolecular probes in a sample, said method comprising thestep of using a device according to claim 6 to detect specific bindingof the biomolecular probes to the immobilized targets.
 32. A method fordetecting biomolecular probes in a sample, said method comprising thestep of using a device according to claim 16 to detect specific bindingof the biomolecular probes to the immobilized targets.
 33. A method fordetecting biomolecular probes in a sample, said method comprising thestep of using a device according to claim 17 to detect specific bindingof the biomolecular probes to the immobilized targets.
 34. A method fordetecting biomolecular probes in a sample, said method comprising thestep of using a device according to claim 18 to detect specific bindingof the biomolecular probes to the immobilized targets.
 35. A method fordetecting biomolecular probes in a sample, said method comprising thestep of using a device according to claim 19 to detect specific bindingof the biomolecular probes to the immobilized targets.
 36. A method fordetecting biomolecular probes in a sample, said method comprising thestep of using a device according to claim 20 to detect specific bindingof the biomolecular probes to the immobilized targets.
 37. A method fordetecting biomolecular probes in a sample, said method comprising thestep of using a device according to claim 21 to detect specific bindingof the biomolecular probes to the immobilized targets.
 38. A method fordetecting biomolecular probes in a sample, said method comprising thestep of using a device according to claim 22 to detect specific bindingof the biomolecular probes to the immobilized targets.
 39. A method fordetecting biomolecular probes in a sample, said method comprising thestep of using a device according to claim 23 to detect specific bindingof the biomolecular probes to the immobilized targets.
 40. A method fordetecting biomolecular probes in a sample, said method comprising thestep of using a device according to claim 24 to detect specific bindingof the biomolecular probes to the immobilized targets.
 41. A method fordetecting biomolecular probes in a sample, said method comprising thestep of using a device according to claim 25 to detect specific bindingof the biomolecular probes to the immobilized targets.